Catabolite control protein C (CcpC) contributes to virulence and hydrogen peroxide-induced oxidative stress responses in Listeria monocytogenes

Listeria monocytogenes causes listeriosis, an infectious and potentially fatal disease of animals and humans. A diverse network of transcriptional regulators, including LysR-type catabolite control protein C (CcpC), is critical for the survival of L. monocytogenes and its ability to transition into the host environment. In this study, we explored the physiological and genetic consequences of deleting ccpC and the effects of such deletion on the ability of L. monocytogenes to cause disease. We found that ccpC deletion did not impact hemolytic activity, whereas it resulted in significant reductions in phospholipase activities. Western blotting revealed that the ?ccpC strain produced significantly reduced levels of the cholesterol-dependent cytolysin LLO relative to the wildtype F2365 strain. However, the ?ccpC mutant displayed no significant intracellular growth defect in macrophages. Furthermore, ?ccpC strain exhibited reduction in plaque numbers in fibroblasts compared to F2365, but plaque size was not significantly affected by ccpC deletion. In a murine model system, the ?ccpC strain exhibited a significantly reduced bacterial burden in the liver and spleen compared to the wildtype F2365 strain. Interestingly, the deletion of this gene also enhanced the survival of L. monocytogenes under conditions of H2O2-induced oxidative stress. Transcriptomic analyses performed under H2O2-induced oxidative stress conditions revealed that DNA repair, cellular responses to DNA damage and stress, metalloregulatory proteins, and genes involved in the biosynthesis of peptidoglycan and teichoic acids were significantly induced in the ccpC deletion strain relative to F2365. In contrast, genes encoding internalin, 1-phosphatidylinositol phosphodiesterase, and genes associated with sugar-specific phosphotransferase system components, porphyrin, branched-chain amino acids, and pentose phosphate pathway were significantly downregulated in the ccpC deletion strain relative to F2365. This finding highlights CcpC as a key factor that regulates L. monocytogenes physiology and responses to oxidative stress by controlling the expression of important metabolic pathways. Overall design: To further investigate the mechanism underlying the reduced susceptibility of the ?ccpC strain to oxidative stress conditions, we examined the regulatory role of CcpC following exposure to H2O2-induced oxidative stress. RNA-seq was performed on total RNA harvested from wildtype and ?ccpC cultures that had been grown to exponential phase and then exposed to 8 mM H2O2 for 2.5 d1031 is the ccpC mutant strain while d1759 is a gltC mutant strain

单核细胞增生李斯特菌（Listeria monocytogenes）可引发李斯特菌病（listeriosis），这是一类能感染动物与人类且具有潜在致死风险的传染病。包括LysR型分解代谢物控制蛋白C（LysR-type catabolite control protein C, CcpC）在内的多样化转录调控网络，对于单核细胞增生李斯特菌的存活以及其向宿主环境定植的能力至关重要。本研究探究了敲除ccpC基因所带来的生理与遗传效应，以及该基因缺失对单核细胞增生李斯特菌致病能力的影响。

研究结果显示，ccpC基因缺失不会影响菌株的溶血活性，但会显著降低其磷脂酶活性。蛋白质免疫印迹（Western blotting）实验表明，相较于野生型菌株F2365，ΔccpC菌株的胆固醇依赖性溶细胞素LLO（cholesterol-dependent cytolysin LLO）的表达量显著下调。不过，ΔccpC突变株在巨噬细胞内的增殖并未出现明显缺陷。此外，相较于F2365菌株，ΔccpC菌株在成纤维细胞中的噬斑数量有所减少，但噬斑大小并未受ccpC基因缺失的显著影响。在小鼠模型体系中，ΔccpC菌株在肝脏与脾脏中的细菌载量显著低于野生型F2365菌株。

值得注意的是，该基因的缺失还能增强单核细胞增生李斯特菌在H₂O₂诱导的氧化应激条件下的存活能力。在H₂O₂诱导的氧化应激条件下开展的转录组学分析显示，相较于F2365菌株，ΔccpC菌株中DNA修复、DNA损伤与应激的细胞应答、金属调控蛋白，以及参与肽聚糖（peptidoglycan）和磷壁酸（teichoic acids）生物合成的基因均被显著诱导表达。与之相反，编码内化素（internalin）、1-磷脂酰肌醇磷酸二酯酶（1-phosphatidylinositol phosphodiesterase）的基因，以及与糖特异性磷酸转移酶系统（phosphotransferase system）组分、卟啉、支链氨基酸（branched-chain amino acids）和戊糖磷酸途径（pentose phosphate pathway）相关的基因，在ΔccpC菌株中均显著下调表达。这一发现表明，CcpC作为关键调控因子，通过控制重要代谢通路的基因表达，调控单核细胞增生李斯特菌的生理状态以及其对氧化应激的应答反应。

整体实验设计：为进一步探究ΔccpC菌株对氧化应激条件耐受性增强的潜在机制，我们检测了CcpC在暴露于H₂O₂诱导的氧化应激后的调控作用。对培养至指数生长期、随后经8 mM H₂O₂处理2.5小时的野生型与ΔccpC菌株的总RNA进行了RNA测序（RNA-seq）。注：d1031为ccpC突变菌株，d1759为gltC突变菌株。